ABSTRACT Tuberculosis (TB), caused by the bacterial pathogen Mycobacterium tuberculosis (Mtb), remains a leading cause of death in the world today despite the availability of therapeutic drugs for more than sixty years. The disease also exacts a significant economic toll in countries where it is prevalent, often in poorer areas of the world. Multidrug-resistant TB (MDR-TB and XDR-TB) is an increasing problem, with nearly one half million cases worldwide per year, adding to the difficulty of controlling this disease. Therefore, effective new drugs, with novel mechanisms of action that can be included in treatment regimens for both drug-susceptible and MDR-TB are urgently needed. Spero Therapeutics has in- licensed SPR720, formerly Vertex VXc-486, an inhibitor of the B subunit (GyrB) ATPase activity of the essential DNA replication gyrase enzyme, that possesses excellent in vitro anti-mycobacterial activity and in vivo efficacy in mouse models of TB infection. GyrB ATPase represents a relatively nave drug target and, therefore, pre-existing resistance to drugs against this target should be rare in patients. The scope of this application includes additional mouse infection model studies to assess treatment efficacy when SPR720 is included in drug regimens that contain an increased dose level of rifampin for drug-susceptible TB and in combinations with existing standard-of-care drugs used for MDR-TB. For the former, a goal in this proposal is to shorten the duration of treatment while for MDR-TB, goals are to identify new drugs to replace those to which isolates are no longer susceptible and also hopefully shorten duration of treatment. Additional profiling of SPR720 indicated favorable drug-like properties including good pharmacokinetics (PK) in multiple animal species, no significant off-target or cytotoxic findings, and no significant toxicities in non-GLP studies in two animal species. In this proposal, we will also work to improve the oral drug formulation as well as assess toxicity in a six-month rat GLP toxicity model in anticipation of future human dosing regimens. In parallel, outside of the scope of this proposal, Spero will run additional studies to complete a full IND-enabling package with an IND filing planned in 2017. Based on the microbiology results and other strong preclinical data to date, Spero believes that this compound is poised for rapid progression into the clinic as a new agent to treat mycobacterial infections and is committed to progressing this compound through full clinical development and entry into the marketplace.